The fundamental organizing principle of the auditory system is tonotopy, whereby sensitivity to frequencies is smoothly mapped across the neurons of each auditory cell group. Place codes, such as tonotopy, are build upon precise pathfinding and neural targeting by individual growth cones. An ideal projection in which to study axonal pathfinding and the tonotopic organization of neural circuitry is that between globular bushy cells of the ventral cochlear nucleus and principal cells of the contralateral medial nucleus of the trapezoid body (MNTB). This project is experimentally accessible, and the genetic program that establishes this neural connection is likely to involve multiple signaling steps, which all can be analyzed in a single coronal slice through the auditory brainstem. These steps include attraction to and cross over the ventral midline, calyceal synapse formation onto specific MNTB principal cell targets. Molecular factors governing axon guidance and targeting in other systems have been examined, but there has been no molecular analysis of projections in the auditory system. In our experimental design, we will work our way across the brainstem, much as the globular bushy cell growth cone does, analyzing the role of signaling proteins and their receptors in guiding axon projection. In Specific Aim 1, we will determine the role of netrin and DCC in directing globular bushy cell axons of the ventral midline. Once at the midline, growth cones must overcome the repulsive action of slit-robo interactions. The role of these two proteins in controlling midline crossing and collateral branching in the auditory system will be analyzed in Specific Aim 2. In Specific Aim 3, we will identify molecules expressed in the developing MNTB or ventral cochlear nucleus that are capable of directing topographic projections of globular bushy cells into specific frequency regions of the MNTB. In Specific Aim 3, we will identify molecules expressed in the developing MNTB or ventral cochlear nucleus that are capable of directing topographic projections of globular bushy cells into specific frequency regions of the MNTB. In Specific Aim 4, we will investigate molecules expressed in the developing MNTB principal cells and globular bushy cells that are capable of directing and stabilizing specific synaptic contacts. The proposed research is significant because it is expected to provide knowledge of molecular guidance cues capable of directing tonotopic organization. This knowledge may generalize to other neural systems.